Nanoparticle-serialized oligonucleotide methods, compositions, and articles

ABSTRACT

The disclosure relates to the use of nanoparticles that are coated with unique oligonucleotide (e.g., DNA) sequences of various base lengths (“nano-DNA”) that act as barcodes for product authentication, product serialization, brand protection, track-and-trace, intelligent supply chain, and law enforcement. The nano-DNA can be incorporated into inks, dyes, resins, labels, and other markings at all manufacturing levels, including the product (unit) level, to encode company and product-specific information. The nano-DNA can also be embedded in the product itself during the manufacturing process. Furthermore, the nano-DNA can be quickly, simply, and inexpensively monitored and verified using an electrochemical biosensor device in resource-limited field conditions.

CROSS REFERENCE TO RELATED APPLICATIONS

Priority is claimed to U.S. Provisional Application Nos. 61/762,618(filed Feb. 8, 2013) and 61/763,142 (filed Feb. 11, 2013), each of whichis incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT INTEREST

None.

BACKGROUND OF THE DISCLOSURE

Field of the Disclosure

The disclosure relates to oligonucleotide (e.g., DNA)-based signaturetechnology incorporating nanoparticle label and separation/recoverymoieties which can be monitored quickly, simply, and inexpensively underresource-limited field settings.

Brief Description of Related Technology

Product counterfeiting is a serious global challenge for legitimatemanufacturers and customers. A flood of sham products undermines thesoftware, computer hardware, pharmaceutical, food, entertainment, andfashion industries—everything from fake designer jeans to phonyprescription drugs. It is estimated that counterfeit products accountfor about 5-7% of world trade, worth an estimated US$600 billion a year.Counterfeiting is a rapidly growing business. It is a serious threat topublic safety, equity, revenue, job markets, and taxes around the world.For example, counterfeit medicines usually do not contain theappropriate active ingredient, causing more harm to the patient andallowing microbes to develop drug resistance. Knock-off toys are not upto code against choking hazards or paint toxicity. Thus, the impact ofproduct counterfeiting is long-term, subtle and diffuse.

Because counterfeit electronic parts adversely affect safety, posesignificant risks to the U.S. government defense supply chain, and driveup costs, the Pentagon's Defense Logistics Agency (DLA) has enacted aDNA-marking requirement of all items falling within Federal Supply Class5962 (Electronic Microcircuits) requiring all such electronicmicrocircuits to include a contractor-unique DNA-based signature.

SUMMARY

The disclosure relates to the use of nanoparticles that are coated withunique oligonucleotide (e.g., DNA) sequences of various base lengths(“nano-DNA”) that act as barcodes for product authentication, productserialization, brand protection, track-and-trace, intelligent supplychain, and law enforcement. The nano-DNA can be incorporated into inks,dyes, resins, labels, and other markings at all manufacturing levels,including the product (unit) level, to encode company andproduct-specific information. The nano-DNA can also be embedded in theproduct itself during the manufacturing process. Furthermore, thenano-DNA can be quickly, simply, and inexpensively monitored andverified using an electrochemical biosensor device in resource-limitedfield conditions.

The disclosed DNA-based anti-counterfeiting technology can be uniquelydesigned for each company and for each product. A nanoparticle coatedwith a DNA sequence having a base length of N will generate N factorial(N!) unique codes. For example, a DNA sequence of base length 10 willgenerate 3.6 million unique codes (10!); a DNA base length of 20 willgenerate up to 20! or 2.4×10¹⁸ (2.4 quintillion) unique codes. Thelength of a serialized oligonucleotide marker and the correspondingnumber unique codes can be uniquely selected for any given company,manufacturing facility, product type, product lot, product serialnumber. Such anti-counterfeiting technology is very difficult to copy asit is uniquely different for each company or product in terms ofsequence and base length. Furthermore, the technology can be read simplyand inexpensively with an electrochemical biosensor, thus allowing forfield-operability, quick results, and inexpensive operation.

By comparison, other DNA-based anti-counterfeiting technologies requireexpensive PCR machines and highly skilled personnel to operate. Thus,the use of such technologies has been limited to major companies thatcan afford to maintain DNA-lab facilities or pay for expensivescreening.

In contrast, the disclosed anti-counterfeiting approach is inexpensive,quick, and simple, thus allowing for more frequent screening of productsat very affordable cost. The technology can be used to protectpharmaceutical products, imported foods, electronics, currency, creditcards, passports, and many other legal documents and goods (e.g.,incorporated into the product itself, incorporated into the productpackaging, printed onto the product or product packaging).

In one aspect, the disclosure relates to a method of identifying anarticle of manufacture, the method comprising: (a) providing an articlecomprising a first serialized oligonucleotide-nanoparticle comprising afirst nanoparticle core and at least one first serializedoligonucleotide (e.g., single-stranded oligonucleotide) attachedthereto, wherein the first serialized oligonucleotide has anoligonucleotide base sequence corresponding to a unique preselected codedefining one or more identifying indicia of the article; (b) providing asecond serialized oligonucleotide-nanoparticle comprising a secondnanoparticle core and at least one second serialized oligonucleotide(e.g., single-stranded oligonucleotide) attached thereto, wherein thesecond serialized oligonucleotide has an oligonucleotide base sequenceat least partially complementary to the first serialized oligonucleotideand capable of hybridizing thereto; (c) combining (i) at least one ofthe article and a sample thereof containing the first serializedoligonucleotide-nanoparticle and (ii) the second serializedoligonucleotide-nanoparticle under conditions sufficient forhybridization between the first serialized oligonucleotide and thesecond serialized oligonucleotide, thereby forming a nanoparticlecomplex comprising the first nanoparticle core attached to the secondnanoparticle core via the hybridized first and second serializedoligonucleotides; (d) separating the nanoparticle complex from thearticle or the sample components remaining after nanoparticle complexformation, thereby forming a purified nanoparticle complex; and (e)electrochemically detecting the at least one of the first nanoparticlecore and the second nanoparticle core in the purified nanoparticlecomplex, thereby determining at least one of the identifying indiciacorresponding to the preselected code of the first serializedoligonucleotide.

In another aspect, the disclosure relates to kit (or system) foridentifying an article of manufacture, the method comprising, the kit(or system) comprising: (a) a first serializedoligonucleotide-nanoparticle comprising a first nanoparticle core and atleast one first serialized oligonucleotide attached thereto, wherein thefirst serialized oligonucleotide has an oligonucleotide base sequencecorresponding to a unique preselected code defining one or moreidentifying indicia of an article; (b) a second serializedoligonucleotide-nanoparticle comprising a second nanoparticle core andat least one second serialized oligonucleotide attached thereto, whereinthe second serialized oligonucleotide has an oligonucleotide basesequence at least partially complementary to the first serializedoligonucleotide and capable of hybridizing thereto; and (c) optionally adatabase comprising the first serialized oligonucleotide base sequence,the unique preselected code and identifying indicia associatedtherewith, the second serialized oligonucleotide base sequence, and theunique preselected code and identifying indicia associated therewith(e.g., electronic database stored in a computer-readable medium;codes/indicia can be the same or different such as one information setis a subset of the other information set).

In another aspect, the disclosure relates to a method for making ortesting an identifiable article of manufacture, the method comprising:(a) providing a kit or system for identifying an article of manufactureaccording to any of the disclosed embodiments; (b) affixing the firstserialized oligonucleotide-nanoparticle to an article (e.g., the articleas the product itself or the article as a label/packaging for theproduct); and (c) releasing the article into a commercial stream at afirst point. In an extension, the method further comprises (d)recovering the article from the commercial stream at a second point(different from the first point); (e) combining (i) at least one of thearticle and a sample thereof containing the first serializedoligonucleotide-nanoparticle and (ii) the second serializedoligonucleotide-nanoparticle under conditions sufficient forhybridization between the first serialized oligonucleotide and thesecond serialized oligonucleotide, thereby forming a nanoparticlecomplex comprising the first nanoparticle core attached to the secondnanoparticle core via the hybridized first and second serializedoligonucleotides; (f) separating the nanoparticle complex from thearticle or the sample components remaining after nanoparticle complexformation, thereby forming a purified nanoparticle complex; and (g)electrochemically detecting the at least one of the first nanoparticlecore and the second nanoparticle core in the purified nanoparticlecomplex, thereby determining at least one of the identifying indiciacorresponding to the preselected code of the first serializedoligonucleotide (e.g., by looking up the preselected code or portionthereof of the first serialized oligonucleotide in the electronicdatabase).

Various refinements and extensions of the disclosed methods, kits,systems, and associated compositions (e.g., serialized oligonucleotidecompositions and related complexes) are possible. For example, (i) thefirst nanoparticle core comprises a metal (e.g., a nanoparticle formedentirely from one or more metals, a nanoparticle core having one or moremetal components, where the metal/metal component is suitable forelectrochemical detection); and/or (ii) the second nanoparticle corecomprises a magnetic material (e.g., suitable for magnetic separation ofthe nanoparticle complex from a sample medium). Similarly, (i) the firstnanoparticle core comprises a magnetic material; and/or (ii) the secondnanoparticle core comprises a metal. In any embodiment, the magneticmaterial can be selected from the group consisting of magnetic ironoxides (e.g., Fe₂O₃, Fe₃O₄; such as nanoparticles formed from themagnetic material alone or combined with another non-magnetic material,for example a non-magnetic metal, a conductive polymer, or anon-conductive polymer (in a magnetic core-non-magnetic shellconfiguration or otherwise)). In any embodiment, the metal can selectedfrom the group consisting of lead, cadmium, zinc, copper, tin, gold,silver, platinum, palladium, ruthenium, rhodium, osmium, iridium,composites thereof, alloys thereof, salts thereof (e.g., sulfates,sulfites, sulfides, chlorides, other halide salts, phosphates, nitrates,nitrites), and combinations thereof. In various refinements, the firstnanoparticle core and the second nanoparticle core each independentlyhave a size of at least 1 nm, 2 nm, 5 nm, 10 nm, 20 nm, 50 nm, or 100 nmand/or up to 10 nm, 20 nm, 50 nm, 100 nm, 200 nm, 500 nm, or 1000 nm(e.g., diameter or other characteristic dimension; average size such asweight-, number-, or volume-average of a distribution of nanoparticles;can apply to metal and/or magnetic nanoparticle cores).

In further refinements, the first serialized oligonucleotide and thesecond serialized oligonucleotide each independently have at least 5,10, 15, 20, 30, or 40 bases and/or up to 10, 15, 20, 30, 40, 60, or 100bases forming their respective oligonucleotide base sequences. In anembodiment, the first serialized oligonucleotide and the secondserialized oligonucleotide have the same coding lengths (e.g., oroverall length; coding length reflects the number of bases correspondingto the preselected code/identifying information is the same for bothserialized oligonucleotides, although additional non-coding bases may bepresent in either or both serialized oligonucleotides for othernon-coding/identification purposes, such that coding lengths are thesame but overall lengths may be different; such as when they both definethe same code/identifying information). In another embodiment, the firstserialized oligonucleotide and the second serialized oligonucleotidehave different coding lengths (e.g., such as when one sequence defines acode/identifying information that is a subset of the other sequenceinformation).

In another refinement, (i) the article comprises a plurality of firstserialized oligonucleotide-nanoparticles; (ii) each first nanoparticlecore comprises a metal; and (iii) the plurality of first serializedoligonucleotide-nanoparticles comprises a plurality of different metalsin a preselected ratio corresponding to an additional preselected codedefining one or more additional identifying indicia of the article(e.g., each nanoparticle core contains a single metal or a combinationof metals, where the combination/ratio of metals in the plurality as awhole defines a characteristic spectrum that can be electrochemicallydetected to determine/differentiate other spectra).

In another refinement, the detection/testing methods further comprisesperforming at least one of PCR analysis or DNA sequencing analysis onthe purified nanoparticle complex to determine all or a portion of theoligonucleotide base sequence of at least one of the first serializedoligonucleotide and the second serialized oligonucleotide (e.g., as aconfirmation of the electrochemical detection and/or to provide furtheridentification, such as when the electrochemical detection identifiesonly a subset of information associated with the first serializedoligonucleotide). In an alternative embodiment, PCR and/or DNAsequencing analysis can be used instead of electrochemical detection ofthe nanoparticle complex (e.g., only the first serializedoligonucleotide-nanoparticle is recovered/analyzed from the article, andthe second serialized oligonucleotide-nanoparticle is not a requiredcomponent of the method or related compositions, kits, systems, etc.).

In further refinements, the article is selected from the groupconsisting of electronic/computer hardware, software encoded in atangible medium, a pharmaceutical composition, a medical device, alegal/financial instrument (e.g., paper or coin currency, credit card orelectronically encoded card such as for performing a financialtransaction), a food item, and packaging for a product (e.g., theforegoing articles or otherwise as products; container, label, box,paper, cardboard, plastic packaging, etc.). In another refinement, thefirst serialized oligonucleotide-nanoparticle is incorporated into thearticle in at least one medium selected from the group consisting of an(invisible or visible) ink applied to the article or a packagingmaterial therefor, a label applied to the article or a packagingmaterial therefor, and a resin or polymer medium as a component of orapplied to the article or a packaging material therefor. In anotherrefinement, the identifying indicia are selected from the groupconsisting of source information, supply chain information, productinformation, and combinations thereof (e.g., any or all ofcompany/supplier name/address, production facility, shipping facility,product information such as product code, lot, facility, unit identifier(e.g., serial number for an individual unit item), dates associated withany production/shipping events, etc.; both serialized oligonucleotidescan encode the same identifying indicia (e.g., as complete complementsof each other) or one serialized oligonucleotide can encode a subset ofinformation from the other serialized oligonucleotide).

More generally, the methods herein can be applied to the testing of anarticle suspected of containing the first serializedoligonucleotide-nanoparticle. In this way, a negative result for theelectrochemical detection of the first or second nanoparticle core canbe used to identify the article as non-authentic/counterfeit. A positiveresult can confirm or suggest the article's authenticity (e.g., subjectto further testing such as PCR or sequencing). Additionally, a positiveresult can be used to trace the history of a product, for example wherethe authenticity of an article is not necessarily in question, but whereit is desirable to know the particular article's history in the streamof commerce.

Additional features of the disclosure may become apparent to thoseskilled in the art from a review of the following detailed description,taken in conjunction with the drawings, examples, and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the disclosure, reference should bemade to the following detailed description and accompanying drawingswherein:

FIG. 1 illustrates a schematic of a nano-DNA detection process accordingto the disclosure.

FIG. 1A illustrates a product or article including a serializedoligonucleotide-nanoparticle label/tracer according to the disclosure.

FIG. 1B illustrates a sampling process for a labeled product or articleto form a detectable nanoparticle complex according to the disclosure.

FIG. 2 is a TEM image of dextrin gold nanoparticles having an averagediameter of about 15 nm.

FIG. 3 is a graph illustrating the UV-vis absorption of dextrin goldnanoparticles at 520 nm wavelength.

FIG. 4 is a graph illustrating the differential pulse voltammetricdetection of different concentrations of nano-DNA under wet or dryconditions.

FIGS. 5A-5C illustrate various embodiments of first and secondserialized oligonucleotide-nanoparticles and related complexes accordingto the disclosure.

While the disclosed compositions, articles, and methods are susceptibleof embodiments in various forms, specific embodiments of the disclosureare illustrated in the drawings (and will hereafter be described) withthe understanding that the disclosure is intended to be illustrative,and is not intended to limit the claims to the specific embodimentsdescribed and illustrated herein.

DETAILED DESCRIPTION

The principle behind the nano-DNA technology is presented in FIG. 1. Thenano-DNA 10 (e.g., gold nanoparticles 12 conjugated with serializedoligonucleotides 14) could be incorporated in product (or article) 70markings, packages, or embedded in the product 70 itself at a specificlocation 72 (FIGS. 1 and 1A). During authentication, a sample 74 of theproduct 70 containing the nano-DNA 10 is removed and hybridized with acomplementary nano-DNA 20 (e.g., magnetic nanoparticles 22 conjugated toserialized oligonucleotides 24, at least a portion of which arecomplementary to those of the serialized oligonucleotides 14 of thenano-DNA 10) to form a two-component nano-DNA conjugate/complex 30(FIGS. 1 and 1B). For example, the nano-DNA 10 (e.g., from the product70 or sample 74 thereof) and the nano-DNA 20 can be added to an aqueousmedium (e.g., a physiological buffer) under time and temperatureconditions suitable for hybridization of the serialized oligonucleotides14, 24 to form the complex 30. The complex 30 can be separated from theaqueous medium (e.g., magnetically separated from other product70/sample 74 components with a magnet 40 when at least one of thenanoparticles 12, 22 is magnetic/magnetically attractable), washed, andthen prepared for further analysis (e.g., prepared as a concentratedsuspension). The specific method for detecting the nano-DNAconjugate/complex 30 (e.g., detecting the either or both of thenanoparticle cores 12, 22 thereof, electrochemically or otherwise) isnot particularly limited. In an illustrative electrochemical detectionmethod, the nano-DNA complex 30 is then added to a microfluidic chip 50that is connected to an electrochemical reader 60. The microfluidic chip50 will process the sample internally and the final solution is flowedinto an electrode chip (e.g., including acidic dissolution of thecomplex 30 to form metal ions corresponding to the metal nanoparticles12, 22 of the nano-DNA 10 and/or 20 (e.g., a metallic component of thefirst and/or second nanoparticle cores 12, 22 thereof); application ofsame to a working surface/electrode of a chip 50 such as an SPCE). Uponapplication of a voltage to the chip 50, the electrochemical reactionoccurs and a signal, in the form of an electric current, is generated.This electrical signal reports the presence of the nano-DNA 10 and/or 20signature (e.g., confirming the product's authenticity when present).

The gold nanoparticles 12 can be replaced with any metallicnanoparticles and their related forms, such as silver (Ag), lead (Pb),lead sulfide (PbS), cadmium (Cd), cadmium sulfide (CdS), zinc (Zn), zincsulfide (ZnS), etc. The magnetic nanoparticles 22 can be replaced withFe₂O₃, Fe₃O₄, magnetic-polyaniline, magnetic-polypyrrole,magnetic-silica, etc. The electrochemical reader 60 can be in any formand size such as handheld, benchtop, etc.

EXAMPLE

The following example illustrates the disclosed processes andcompositions, but is not intended to limit the scope of any claimsthereto.

Gold nanoparticles (AuNPs) were synthesized under alkaline conditionfollowing the approach published by Anderson et al (2011). Briefly, 20ml of dextrin stock solution (25 g/l) and 20 ml of sterile water weremixed in a 50 ml sterile orange cap tube (disposable). Five millilitersof HAuCl₄ stock solution (8 g/ml) were then added, and the pH of thesolution was adjusted to 9 with sterile 10% (w/v) Na₂CO₃ solution. Thefinal volume was brought to 50 ml with pH 9 water. The reaction wascarried out by incubating the solution in a sterile flask in the dark at50° C. with continuous shaking (100 rpm) for 6 h. A red solution wasobtained at the end of the reaction. The final concentration of AuNP was10 mg/ml. FIG. 2 shows a transmission electron microscope (TEM) image ofgold nanoparticles with about 15 nm in diameter. They have beencharacterized for UV-vis absorption at 520 nm (FIG. 3) and for theirelectrochemical properties.

The AuNPs were coated with synthetic oligonucleotides (sequence lengthmay vary, preferably from 5 to 30 bases). Briefly, thiolatedoligonucleotides were mixed with 100 μl of 0.1 M DTT solution and thenpurified using a Nap-5 column. The oligonucleotides (5 nmol) were addedto 1 ml of AuNPs and a serial salt addition was conducted for 3 h. Thethiolated DNA would form a self-assembled monolayer on the surface ofAuNPs. After washing away the excess reagents, the AuNP-DNA conjugates(or nano-DNA) were ready for use and could be stored at roomtemperature.

The foregoing procedure was used to attach two validationoligonucleotide sequences to the AuNPs:5-AATATGCTGCCTACTGCCCTACGCTT-5H-3′ (SEQ ID NO:1; 26 bases) and5-AGATTTAAATCTGGTAGAAAGGCGG-5H-3′ (SEQ IS NO: 2; 25 bases). Bothsequences could be detected using AuNPs as electrochemical reporters asshown in FIG. 4 (differential pulse voltammogram of different nano-DNAconcentrations on screen carbon printed electrodes (SPCE); D-1=1:10dilution, D-2=1:100 dilution, D-3=1:1000 dilution). Wet samples weredetected immediately with minimum or no accumulation time. The AuNPdissolution with HCl was performed in liquid before the sample wastransferred to the SPCE. Dry samples were deposited on the SPCE and theHCl was applied after the solution was completely dry.

Conjugation of magnetic nanoparticles (MNPs) with DNA probes (havingmatching/complementary sequences to the oligonucleotides conjugated tothe AuNPs) could be performed in various ways depending on the type ofmagnetic nanoparticles used. One way is to use polyamine-functionalizediron oxide (PIO) particles. In this method (Zhang et al., 2009; Zhang etal., 2010), 1 mg of PIO were reacted with 300 μg of sulfo-SMCCbifunctional linker for 2 h in 1 mL coupling buffer (0.1 M PBS buffer,0.2M NaCl, pH 7.2). After rinsing, the reduced thiolated DNA probe (1nmol) was added into 1 mL coupling buffer containing sulfo-SMCC-modifiedMNPs and reacted for 8 h. The functionalized MNPs were then suspended in35 mL of 10 mM sulfo-NHS acetate. The solution was incubated and shakenat room temperature to block the unreacted sulfo-SMCC on the surface ofMNPs. After passivation, the particles were centrifuged at 4000 rpm for1 min and washed with passivation buffer (0.2M Tris, pH 8.5) and thenwith a storage buffer (10 mM PBS buffer, 0.2M NaCl, pH 7.4). TheMNP-probe conjugates were stored at 4° C. before use.

FIGS. 5A-5C illustrate various embodiments of first and secondserialized oligonucleotide-nanoparticles and related complexes accordingto the disclosure. In FIG. 5A, a nanoparticle complex 30 includes afirst serialized oligonucleotide-nanoparticle 10 and a second serializedoligonucleotide-nanoparticle 20. The first serializedoligonucleotide-nanoparticle 10 includes a first nanoparticle core 12and a first serialized oligonucleotide 14 attached thereto (illustratedas a single oligonucleotide 14, although the core 12 can have aplurality of attached oligonucleotides 14). Similarly, the secondserialized oligonucleotide-nanoparticle 20 includes a secondnanoparticle core 22 and a second serialized oligonucleotide 24 attachedthereto (illustrated as a single oligonucleotide 24, although the core22 can have a plurality of attached oligonucleotides 24). The first andsecond serialized oligonucleotides 14, 24 are hybridized together,linking the nanoparticle cores 12, 22 and forming the complex 30 (e.g.,as originally formed in a sample analysis or sample matrix, or assubsequently purified from a sample matrix for electrochemicaldetection).

FIG. 5B illustrates an embodiment in which the first serializedoligonucleotide-nanoparticle composition 10 incorporated into an article70 includes a plurality of first serializedoligonucleotide-nanoparticles 10 containing a plurality of differentmetal nanoparticle cores 12 _(i) (e.g., for i=1 to n differentmetals/metal combinations). The different metal nanoparticle cores 12_(i) are incorporated into an article 70 in a preselected ratiocorresponding to an additional preselected code defining one or moreadditional identifying indicia of the article. As an illustration, agiven set of 3 different metal nanoparticle cores 12 ₁, 12 ₂, and 12 ₃having the same first serialized oligonucleotide 14 may be incorporatedinto different articles at ratios of 1:1:1 and 1:2:1 (for example). Byelectrochemically resolving the differing ratios, the predeterminedplurality of different metal nanoparticle cores 12 _(i) andcorresponding ratios can be used to extend the coding informationcontained in the first serialized oligonucleotide 14.

FIG. 5C illustrates an embodiment in which the first serializedoligonucleotide 14 and the second serialized oligonucleotide 24 havedifferent coding lengths (e.g., such as when one sequence defines acode/identifying information that is a subset of the other sequenceinformation). For example, the first serialized oligonucleotide 14 canbe represented by a series of coding regions 14 _(i) (e.g., for i=1 ton), where each region 14 _(i) corresponds to a particular subset of allof the code/information denoted by the oligonucleotide 14. Similarly,the second serialized oligonucleotide 24 can be represented by a seriesof coding regions 24 _(i) (e.g., for i=1 to m, where m<n), where eachregion 24 _(i) corresponds to a particular subset of all of thecode/information denoted by the oligonucleotide 24. For example, asillustrated, regions 14 ₁ and 14 ₂ could represent the article supplierand product code, respectively, while 14 ₃ to 14 _(n) could representmore detailed product information (e.g., lot number, unit-level serialnumber). Similarly, complementary regions 24 ₁ and 24 ₂ also representthe article supplier and product code. In this way, a “universal” secondserialized oligonucleotide-nanoparticle 20 composition can be used torapidly identify top-level information related to a product (e.g.,corresponding to coding regions 1 and/or 2 as illustrated), whilelower-level information can be subsequently determined using PCR and/orsequencing analysis. As illustrated in FIG. 5C, suitably, when oneserialized oligonucleotide is selected to be complementary to asubsequence of the corresponding oligonucleotide, the region forhybridization in the longer base sequence can be selected to be at aterminal region opposite the attachment point of the oligonucleotide tothe nanoparticle core.

Nanoparticles

The type and size of the nanoparticles are not particularly limited. Thenanoparticles can metal nanoparticles, metal-containing nanoparticles,polymer nanoparticles, polymer-containing nanoparticles, magneticnanoparticles, and combinations thereof (e.g., as nanocomposites,core-shell nanoparticles, etc.). In various embodiments, the metal ormetal-containing nanoparticles can include lead, cadmium, zinc, copper,tin, gold, silver, platinum, palladium, ruthenium, rhodium, osmium,iridium, composites thereof, alloys thereof, salts thereof (e.g.,sulfates, sulfites, sulfides, chlorides, other halide salts, phosphates,nitrates, nitrites), and combinations thereof. Suitable metalnanoparticles include gold nanoparticles. Suitable metal-containingnanoparticles include quantum dots (e.g., CdSe, ZnS, CdS, PbS or anothermetal-containing nanoparticle which can provide an electrochemicaldetection signal based on its metal component and/or a fluorescentdetection signal based on its quantum dot nanocrystal structure). Themagnetic nanoparticles can include magnetic iron oxides (e.g., Fe₂O₃,Fe₃O₄), for example nanoparticles formed from the magnetic materialalone or combined with another non-magnetic material (e.g., anon-magnetic metal, conductive polymer, or non-conductive polymer suchas in a magnetic core-non-magnetic shell configuration or otherwise). Invarious embodiments, the first nanoparticle and the second nanoparticlein any of the various forms each independently have a size of at least 1nm, 2 nm, 5 nm, 10 nm, 20 nm, 50 nm, or 100 nm and/or up to 10 nm, 20nm, 50 nm, 100 nm, 200 nm, 500 nm, or 1000 nm (i.e., the first andsecond nanoparticle sizes can be the same or different). Nanoparticlesize can represent nanoparticle diameter or other characteristicdimension, for example an average size such as weight-, number-, orvolume-average of a distribution of nanoparticles, whether for metalnanoparticles, metal-containing nanoparticles, polymer nanoparticles,polymer-containing nanoparticles, magnetic nanoparticles, or otherwise.

Metal nanoparticles may be formed by any method known in the art. By wayof illustration, a suitable method of metal nanoparticle formationincludes reduction of metal ions in an aqueous reaction system furtherincluding a carbohydrate (or other) capping agent. The metal ions in theaqueous medium are reduced at a neutral or alkaline pH value in thepresence of the carbohydrate capping agent under suitable reactionconditions to form reduced metal nanoparticles (e.g., at a reactiontemperature and reaction time sufficient to convert all or substantiallyall of the metal ion precursors). The reaction generally includes aninitial nucleation stage to form metallic nuclei followed by a longergrowth stage in which metal ions reduced on the nuclei surfaces createthe final metal nanoparticles. The reduced metal nanoparticles are inthe form of a stabilized suspension of metal nanoparticles in theaqueous medium, where the carbohydrate capping agent stabilizes theformed nanoparticle suspension.

The specific metal ions or oxidized metal-containing species in solutionand selected as precursors to the desired metal nanoparticles are notparticularly limited and are suitably chosen according to a desired enduse/application of the nanoparticle suspension. In an embodiment, themetal ions include gold ions (e.g., Au(III), Au³⁺) and are selected toform gold metal nanoparticles (AuNPs). The metal ions can be free insolution (e.g., introduced into the aqueous medium as a dissolvableionic compound, for example a salt or acid) or coordinated/coupled withother (ionic) species. Other potential metal ions can include chromium,copper, zinc, nickel, cadmium, silver, cobalt, indium, germanium, tin,lead, arsenic, antimony, bismuth, chromium, molybdenum, manganese, iron,ruthenium, rhodium, palladium, osmium, iridium, and platinum. In someembodiments, two or more types of metal ions can be in solution in theaqueous medium to provide metal nanoparticles formed from alloys of twoor more elemental metals. The concentration of metal ions in solutionprior to reaction is not particularly limited, but it suitably rangesfrom 0.1 mM to 1000 mM (e.g., at least 0.1 mM, 1 mM, or 10 mM and/or upto 100 mM or 1000 mM). The population of the reduced metal nanoparticlesas produced (e.g., in suspension as formed in the aqueous medium orotherwise) generally has a particle size ranging from 2 nm to 50 nm(e.g., a number-, weight-, or volume-average particle size). Forexample, the average size of the metal nanoparticle distribution can beat least 2, 5, 8, 10, 12, or nm and/or up to 8, 10, 12, 15, 20, 25, 30,40, or 50 nm.

In some embodiments, the aqueous medium further includes, prior toreduction of the metal ions, a population of nanoparticles serving ascores/nucleation sites for deposition of the reduced metal ions, thuspermitting the formation of metal nanoparticles having a core-shellstructure including a nanoparticle core with a metallic shell. Thenanoparticle core material is not particularly limited and can benon-metallic, metallic (e.g., different from the metal to be reduced asa shell), magnetic, etc. Magnetic nanoparticle cores are particularlyuseful to permit the resulting metal nanoparticle to function as both amagnetic sample/analyte separator and concentrator (e.g., due to themagnetic core) as well as a signal transducer (e.g., due to theelectrical properties of the metal shell material such as gold).

The magnetic nanoparticles are not particularly limited and generallyinclude any nano-sized particles (e.g., about 1 nm to about 1000 nm)that can be magnetized with an external magnetic/electrical field. Forexample, the magnetic nanoparticles can include superparamagneticparticles, which particles can be easily magnetized with an externalmagnetic field (e.g., to facilitate separation or concentration of theparticles from the bulk of a sample medium) and then redispersedimmediately once the magnet is removed (e.g., in a new (concentrated)sample medium). Thus, the magnetic nanoparticles are generally separablefrom solution with a conventional magnet. Suitable magneticnanoparticles are provided as magnetic fluids or ferrofluids, and mainlyinclude nano-sized iron oxide particles (Fe₃O₄ (magnetite) or γ-Fe₂O₃(maghemite)) suspended in a carrier liquid. Such magnetic nanoparticlescan be prepared by superparamagnetic iron oxide by precipitation offerric and ferrous salts in the presence of sodium hydroxide andsubsequent washing with water. A suitable source of γ-Fe₂O₃ isSigma-Aldrich (St. Louis, Mo.), which is available as a nano-powderhaving particles sized at <50 nm with a specific surface area rangingfrom about 50 m²/g to about 250 m²/g. Preferably, the magneticnanoparticles have a small size distribution (e.g., ranging from about 5nm to about 25 nm) and uniform surface properties (e.g., about 50 m²/gto about 245 m²/g).

More generally, the magnetic nanoparticles can include ferromagneticnanoparticles (i.e., iron-containing particles providing electricalconduction or resistance). Suitable ferromagnetic nanoparticles includeiron-containing magnetic metal oxides, for example those including ironeither as Fe(II), Fe(III), or a mixture of Fe(II)/Fe(III). Non-limitingexamples of such oxides include FeO, γ-Fe₂O₃ (maghemite), and Fe₃O₄(magnetite). The magnetic nanoparticles can also be a mixed metal oxideof the type M1_(x)M2_(3-x)O₄, wherein M1 represents a divalent metal ionand M2 represents a trivalent metal ion. For example, the magneticnanoparticles may be magnetic ferrites of the formula M1Fe₂O₄, whereinM1 represents a divalent ion selected from Mn, Co, Ni, Cu, Zn, or Ba,pure or in admixture with each other or in admixture with ferrous ions.Other metal oxides include aluminum oxide, chromium oxide, copper oxide,manganese oxide, lead oxide, tin oxide, titanium oxide, zinc oxide andzirconium oxide, and suitable metals include Fe, Cr, Ni or magneticalloys.

In some embodiments, the nanoparticles can include a conductive polymer(e.g., as the complete nanoparticle; as a coating or shell for a metaland/or magnetic core in a composite nanoparticle). The conductivepolymers according to the disclosure are not particularly limited andgenerally include any polymer that is electrically conductive.Preferably, the conductive polymer is fluid-mobile when bound to ananalyte. Suitable examples of conductive polymers are polyanilines,polypyrrole, and polythiophenes, which are dispersible in water and areconductive because of the presence of an anion or cation in the polymer(e.g., resulting from acid-doping of the polymer or monomer). Otherelectrically conductive polymers include substituted and unsubstitutedpolyanilines, polyparaphenylenes, polyparaphenylene vinylenes,polythiophenes, polypyrroles, polyfurans, polyselenophenes,polyisothianapthenes, polyphenylene sulfides, polyacetylenes,polypyridyl vinylenes, biomaterials, biopolymers, conductivecarbohydrates, conductive polysaccharides, combinations thereof andblends thereof with other polymers, copolymers of the monomers thereof.Conductive polyanilines are preferred. Polyaniline is perhaps the moststudied conducting polymer in a family that includes polypyrrole,polyacetylene, and polythiophene. As both electrical conductor andorganic compound, polyaniline possesses flexibility, robustness, highlycontrollable chemical and electrical properties, simple synthesis, lowcost, efficient electronic charge transfer, and environmental stability.Addition of a protic solvent such as hydrochloric acid yields aconducting form of polyaniline, with an increase in conductivity of upto ten orders of magnitude. Illustrative are the conductive polymersdescribed in U.S. Pat. Nos. 6,333,425, 6,333,145, 6,331,356 and6,315,926. Preferably, the conductive polymers do not contain metals intheir metallic form.

The conductive polymer provides a substrate for the subsequentattachment of a serialized oligonucleotide bound thereto. Theelectrically conductive characteristics of the conductive polymer alsocan facilitate electrochemical detection of a nanoparticle complexincluding a serialized oligonucleotide-conductive polymer nanoparticle(or conductive polymer-containing nanoparticle), for example bymeasuring the electrical resistance or conductance through a pluralityof conductive polymer nanoparticles immobilized in a capture ordetection region of a conductimetric biosensor device.

Serialized Oligonucleotides

Identification and authentication of an article of manufacture accordingto the disclosure are based on first and second serializedoligonucleotide strands (e.g., single-stranded oligonucleotides or ssDNAstrands) that are designed to at least partially (or completely)hybridize. As known to a person skilled in the art, an ssDNA strandcontains a 5′-to-3′ directionality. Two ssDNA strands will bind (orhybridize) according to the Watson-Crick base paring principle (i.e., anadenine (A) binds to a thymine (T) and a cytosine binds to a guanine(G)). An ssDNA strand or oligonucleotide includes a single free strandof polymerized deoxyribonucleic acids consisting of repeated polymerbases of adenine (A), cytosine (C), guanine (G), and/or thymine (T),where each strand has directionality and runs from five prime (5′) tothree prime (3′). The two strands when bound together have oppositedirectionality (i.e., they run anti-parallel). This A/T and C/G baseparing is highly specific, and the mismatching rates of such basepairing are very low for relatively short ssDNA sequences. Thisprinciple, coupled with preselected sequences for the first and secondserialized oligonucleotides which correspond to a unique preselectedcode/identifying index of an associated article, permits articlelabeling, identification, and/or authentication.

The length and specific sequences for the first serializedoligonucleotide and the second serialized oligonucleotide are notparticularly limited. Relatively longer sequences permit relatively moreencoded information to be represented by the serializedoligonucleotides. As used herein, the encoded information includes thepreselected codes defining the identifying indicia corresponding thereto(e.g., as contrasted to DNA/oligonucleotide sequences in biologicalsystems which encode amino acid sequences or contain other geneticinformation in an organism). Relatively longer sequences also reduce thelikelihood that either of the serialized oligonucleotides will hybridizewith one or more natural DNA/oligonucleotide sequences which may bepresent in a particular sample being tested (e.g., as a result of aparticular product including serialized oligonucleotide-nanoparticlesbeing exposed to the external environment such as in the normal streamof commerce). Suitably, the sequences of the serialized oligonucleotidesare selected to be non-complementary to naturally occurring DNAsequences (e.g., unable to hybridize with naturally occurring DNAsequences or known genomic DNA sequences, as in a silent DNA sequence oroligonucleotide) in addition to encoding identifying information. Eachserialized oligonucleotide independently can be selected to have atleast 5, 10, 15, 20, 30, or 40 bases and/or up to 10, 15, 20, 30, 40,60, or 100 bases forming its respective oligonucleotide base sequences.In an embodiment, the first serialized oligonucleotide and the secondserialized oligonucleotide have the same coding lengths (e.g., oroverall length). The coding length reflects the number of basescorresponding to the preselected code/identifying information and can bethe same for both serialized oligonucleotides, although additionalnon-coding bases may be present in either or both serializedoligonucleotides for other non-coding/identification purposes, such thatcoding lengths are the same but overall lengths may be different (e.g.,when both serialized oligonucleotides define the same code/identifyinginformation). In another embodiment, the first serializedoligonucleotide and the second serialized oligonucleotide have differentcoding lengths (e.g., such as when one sequence defines acode/identifying information that is a subset of the other sequenceinformation).

The first and second serialized oligonucleotides can be produced usingmethods generally known to those skilled in the art. Suitably, theoligonucleotide sequences may be selected to have from 5 bases to 100bases (e.g., or from 5 to 30 bases). The first and second serializedoligonucleotides having the desired, preselected sequences generally canbe custom ordered from commercial sources (e.g., available fromIntegrated DNA Technologies; Coralville, Iowa), and sucholigonucleotides can be selected to include further functionalmodifications (e.g., 5′-end and/or 3′-end modification to facilitateserialized oligonucleotide-nanoparticle attachment and/or (hybridized)serialized oligonucleotide-nanoparticle detection, such as with a thiolfunctional group or fluorescent dye appended to the serializednucleotide).

The first and second serialized oligonucleotides can be attached totheir respective nanoparticle cores to form the corresponding serializedoligonucleotide-nanoparticle structure according to various methodsknown in the art for a desired nanoparticle application. General methodsof oligonucleotide attachment can include physical adsorption (e.g.,resulting from electrostatic (metal) nanoparticle-oligonucleotideinteractions), direct binding (e.g., based on affinity interactionsbetween the (metal) nanoparticle surface and a functional group of theoligonucleotide, such as between a thiolated oligonucleotide and gold),covalent attachment (e.g., between the oligonucleotide and a covalentlinking intermediate that is bound to the (metal) nanoparticle, such asthrough thiolated carboxylic acids, EDAC-mediated or SMCC-mediatedattachment of oligonucleotides, biotin-streptavidin linking, andazide-linking or other “click” functionalization techniques). Forexample, when attaching serialized oligonucleotides to a conductivepolymer-containing nanoparticle (e.g., a magnetic nanoparticle, such asa polyaniline-coated magnetic nanoparticle), oligonucleotides can beincubated in a buffer (e.g., an acetate buffer at a pH of about 5.2)suspension of the nanoparticle that also includes an immunoconjugatingagent (e.g., 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (“EDAC”)). Similarly,sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(“sulfo-SMCC”) is a water-soluble, amine-to-sulfhydryl crosslinkersuitable for providing a covalent linkage between an amine group (e.g.,in a polyamine-functionalized nanoparticle) and a thiol group (e.g., ina thiolated oligonucleotide). In an embodiment, the serializedoligonucleotide can be immobilized on a metal nanoparticle using aligand exchange process known in the art (e.g., when the metalnanoparticle is stabilized with a capping agent). For example, in ageneral ligand exchange process, a carbohydrate capping agentstabilizing the metal nanoparticle suspension is removed from the outersurface of the metal nanoparticles (e.g., partial or complete removal ofthe capping agent). Removal of the capping agent promotes increasedaccess to surface areas of the metal nanoparticles, thus allowingimmobilization of the serialized oligonucleotide on the outer surface ofthe metal nanoparticle (e.g., by contacting/incubating the metalnanoparticle suspension with the serialized oligonucleotide). Forexample, a suitable ligand exchange method for gold nanoparticlesincludes a DTT-mediated removal of a (carbohydrate) capping agentfollowed by immobilization of a thiolated serialized oligonucleotide onthe gold nanoparticle surface. Another suitable ligand exchange methodfor gold nanoparticles includes a surfactant-mediated removal of a(carbohydrate) capping agent followed by immobilization of a thiolatedattachment intermediate (e.g., thiolated carboxylic acid used forfurther covalent attachment of a serialized oligonucleotide) on the goldnanoparticle surface.

The hybridization conditions for forming the nanoparticle complex (e.g.,hybridized conjugate between the first and second serializedoligonucleotide-nanoparticles) are not particularly limited and aregenerally known to the skilled artisan. Suitably, an aqueous mixture orsuspension containing the first serialized oligonucleotide-nanoparticles10 and the second serialized oligonucleotide-nanoparticles 20 (e.g., ina physiological buffer, such as Tris-Saline EDTA at pH 7.4) is heated toa temperature sufficient to initiate hybridization (e.g., at least 25°C., 50° C., or 75° C. and/or up to 95° C.; holding at the maximumheating temperature for 1 min to 5 min) and then allowed to cool (e.g.,to room temperature, such as 20° C. to 25° C.), during which timefurther hybridization occurs.

The various references cited herein and listed below are incorporated byreference in their entireties, in particular in relation to theirdisclosures related to the formation of nanoparticles andnanoparticle-oligonucleotide conjugates (e.g., magnetic nanoparticles,conductive polymer-coated nanoparticles, gold nanoparticles, and/orother metal nanoparticle tracers alone or conjugated with anoligonucleotide) as well as the detection of same (e.g., magneticseparation/concentration, electrochemical detection).

Because other modifications and changes varied to fit particularoperating requirements and environments will be apparent to thoseskilled in the art, the disclosure is not considered limited to theexample chosen for purposes of illustration, and covers all changes andmodifications which do not constitute departures from the true spiritand scope of this disclosure.

Accordingly, the foregoing description is given for clearness ofunderstanding only, and no unnecessary limitations should be understoodtherefrom, as modifications within the scope of the disclosure may beapparent to those having ordinary skill in the art.

All patents, patent applications, government publications, governmentregulations, and literature references cited in this specification arehereby incorporated herein by reference in their entirety. In case ofconflict, the present description, including definitions, will control.

Throughout the specification, where the compositions, processes, kits,or apparatus are described as including components, steps, or materials,it is contemplated that the compositions, processes, or apparatus canalso comprise, consist essentially of, or consist of, any combination ofthe recited components or materials, unless described otherwise.Component concentrations can be expressed in terms of weightconcentrations, unless specifically indicated otherwise. Combinations ofcomponents are contemplated to include homogeneous and/or heterogeneousmixtures, as would be understood by a person of ordinary skill in theart in view of the foregoing disclosure.

REFERENCES

-   1. Alocilja et al. U.S. Pat. No. 8,287,810.-   2. Alocilja et al. U.S. Publication No. 2009/0123939.-   3. Alocilja et al. U.S. Publication No. 2011/0171749.-   4. Alocilja et al. U.S. Publication No. 2012/0315623.-   5. Alocilja et al. U.S. Publication No. 2014/0024026.-   6. Anderson, J. M., Torres-Chavolla, E., Castro, A. B., and    Alocilja, E. C. 2011. One step alkaline synthesis of biocompatible    gold nanoparticles using dextrin as capping agent. Journal of    Nanoparticle Research, 13(7), 2843-2851. DOI    10.1007/s11051-010-0172-3.-   7. Zhang, D., Huang, M. C., and Alocilja, E. C. 2010. A Multiplex    Nanoparticle-based Bio-barcoded DNA Sensor for the Simultaneous    Detection of Multiple Pathogens. Biosensors and Bioelectronics,    26(4), 1736-1742.-   8. Zhang, D., Carr, D. J., and Alocilja, E. C. 2009. Fluorescent    bio-barcode DNA assay for the detection of Salmonella enterica    serovar Enteritidis. Biosensors and Bioelectronics, 24(5),    1377-1381.

What is claimed is:
 1. A method of identifying an article ofmanufacture, the method comprising: (a) providing an article comprisinga plurality of first serialized oligonucleotide-nanoparticles, eachfirst serialized oligonucleotide-nanoparticle comprising (i) a firstnanoparticle core comprising a metal and (ii) at least one firstserialized oligonucleotide attached thereto, wherein the firstserialized oligonucleotide has an oligonucleotide base sequencecorresponding to a unique preselected code defining one or moreidentifying indicia of the article, and the plurality of firstserialized oligonucleotide-nanoparticles comprises a plurality ofdifferent metals in a preselected ratio corresponding to an additionalpreselected code defining one or more additional identifying indicia ofthe article; (b) providing a second serializedoligonucleotide-nanoparticle comprising a second nanoparticle core andat least one second serialized oligonucleotide attached thereto, whereinthe second serialized oligonucleotide has an oligonucleotide basesequence at least partially complementary to the first serializedoligonucleotide and capable of hybridizing thereto; (c) combining (i) atleast one of the article and a sample thereof containing the firstserialized oligonucleotide-nanoparticle and (ii) the second serializedoligonucleotide-nanoparticle under conditions sufficient forhybridization between the first serialized oligonucleotide and thesecond serialized oligonucleotide, thereby forming a nanoparticlecomplex comprising the first nanoparticle core attached to the secondnanoparticle core via the hybridized first and second serializedoligonucleotides; (d) separating the nanoparticle complex from thearticle or the sample components remaining after nanoparticle complexformation, thereby forming a purified nanoparticle complex; and (e)electrochemically detecting the at least one of the first nanoparticlecore and the second nanoparticle core in the purified nanoparticlecomplex, thereby determining at least one of the identifying indiciacorresponding to the preselected code of the first serializedoligonucleotide.
 2. The method of claim 1, wherein: the secondnanoparticle core comprises a magnetic material.
 3. The method of claim2, wherein the magnetic material is selected from the group consistingof magnetic iron oxides.
 4. The method of claim 1, wherein the metal offirst serialized oligonucleotide-nanoparticle is selected from the groupconsisting of lead, cadmium, zinc, copper, tin, gold, silver, platinum,palladium, ruthenium, rhodium, osmium, and iridium.
 5. The method ofclaim 1, wherein: the first nanoparticle core comprises a magneticmaterial.
 6. The method of claim 5, wherein the magnetic material isselected from the group consisting of magnetic iron oxides.
 7. Themethod of claim 1, wherein the second nanoparticle core comprises ametal, wherein the metal is selected from the group consisting of lead,cadmium, zinc, copper, tin, gold, silver, platinum, palladium,ruthenium, rhodium, osmium, and iridium.
 8. The method of claim 1,wherein the first nanoparticle core and the second nanoparticle coreeach independently have a size ranging from 1 nm to 1000 nm.
 9. Themethod of claim 1, wherein the first serialized oligonucleotide and thesecond serialized oligonucleotide each independently have from 5 basesto 100 bases forming their respective oligonucleotide base sequences.10. The method of claim 1, wherein the first serialized oligonucleotideand the second serialized oligonucleotide have the same coding lengths.11. The method of claim 1, wherein the first serialized oligonucleotideand the second serialized oligonucleotide have different coding lengths.12. The method of claim 1, further comprising performing at least one ofPCR analysis or DNA sequencing analysis on the purified nanoparticlecomplex to determine all or a portion of the oligonucleotide basesequence of at least one of the first serialized oligonucleotide and thesecond serialized oligonucleotide.
 13. The method of claim 1, whereinthe article is selected from the group consisting of electronic/computerhardware, software encoded in a tangible medium, a pharmaceuticalcomposition, a medical device, a legal instrument, a financialinstrument, a food item, and packaging for a product.
 14. The method ofclaim 1, wherein the first serialized oligonucleotide-nanoparticle isincorporated into the article in at least one medium selected from thegroup consisting of an ink applied to the article or a packagingmaterial therefor, a label applied to the article or a packagingmaterial therefor, and a resin or polymer medium as a component of orapplied to the article or a packaging material therefor.
 15. The methodof claim 1, wherein the identifying indicia are selected from the groupconsisting of source information, supply chain information, productinformation, and combinations thereof.
 16. The method of claim 1,further comprising, prior to part (c): releasing the article comprisingthe first serialized oligonucleotide-nanoparticle into a commercialstream at a first point; and recovering the article from the commercialstream at a second point.